Cervical cancer screening strategies using visual inspection or cytology may have suboptimal diagnostic accuracy for detection of precancer in women living with HIV (WLHIV). The optimal screen and screen–triage strategy, age to initiate, and frequency of screening for WLHIV remain unclear. This study evaluated the sensitivity, specificity, and positive predictive value of different cervical cancer strategies in WLHIV in Africa.p < 0.001), 28.6% and 49.6% ever had prior cervical cancer screening (p < 0.001), and 69.9% and 64.2% were taking ART at enrolment (p = 0.045) in BF and SA, respectively. CIN2+ prevalence was 5.8% and 22.4% in BF and SA (p < 0.001), respectively. VIA had low sensitivity for CIN2+  and CIN3+  in both countries, with specificity for ≤CIN1 of 78.7% (95% CI 76.0%–81.3%). HC2 had sensitivity of 88.8% (95% CI 82.9%–93.2%) for CIN2+ and 86.4% (95% CI 75.7%–93.6%) for CIN3+. Specificity for ≤CIN1 was 55.4% (95% CI 52.2%–58.6%), and screen positivity was 51.3%. Specificity was higher with a restricted genotype (HPV16/18/31/33/35/45/52/58) approach , with lower screen positivity (33.7%), although there was lower sensitivity for CIN3+ . In BF, HC2 was more sensitive for CIN2+/CIN3+ compared to VIA/VILI . Triage of HC2-positive women with VIA/VILI reduced the number of colposcopy referrals, but with loss in sensitivity for CIN2+ (58.1%) but not for CIN3+ (84.6%). In SA, cytology high-grade squamous intraepithelial lesion or greater (HSIL+) had best combination of sensitivity  and specificity . HC2 had similar sensitivity for CIN3+  but lower specificity compared to HSIL+ , resulting in almost twice as many referrals. Compared to HC2, triage of HC2-positive women with HSIL+ resulted in a 40% reduction in colposcopy referrals but was associated with some loss in sensitivity. CIN2+ incidence over a median 16 months was highest among VIA baseline screen-negative women  and women who were baseline double-negative with HC2 and VIA  and lowest among HC2 baseline screen-negative women . Limitations of our study are that WLHIV included in the study may not reflect a contemporary cohort of WLHIV initiating ART in the universal ART era and that we did not evaluate HPV tests available in study settings today.WLHIV aged 25–50 years attending HIV treatment centres in Burkina Faso (BF) and South Africa (SA) from 5 December 2011 to 30 October 2012 were enrolled in a prospective evaluation study of visual inspection using acetic acid (VIA) or visual inspection using Lugol’s iodine (VILI), high-risk human papillomavirus DNA test (Hybrid Capture 2 [HC2] or careHPV), and cytology for histology-verified high-grade cervical intraepithelial neoplasia (CIN2+/CIN3+) at baseline and endline, a median 16 months later. Among 1,238 women , median age was 36 and 34 years  test targeting 14 high-risk (HR) types had higher sensitivity to detect CIN2+ compared to visual inspection but had low specificity, although a restricted genotype approach targeting 8 HR types decreased the number of unnecessary colposcopy referrals. Cytology HSIL+ had optimal performance for CIN2+/CIN3+ detection in SA. Triage of HPV-positive women with HSIL+ maintained high specificity but with some loss in sensitivity compared to HC2 alone. In this cohort study, Helen Kelly and colleagues explore cervical cancer screening strategies for women living with HIV. Invasive cervical cancer is the second most common cancer among women in low- and middle-income countries and a leading cause of cancer-related death in women in sub-Saharan Africa.Women living with human immunodeficiency virus (WLHIV) have an increased risk of cervical cancer and precancer. The majority of WLHIV live in low- and middle-income countries, where access to cervical cancer screening and treatment of precancerous cervical lesions is limited.Screening approaches most commonly used in sub-Saharan Africa, including visual inspection of the cervix and cervical cytology, which checks for cervical cell abnormalities, have shown variable diagnostic accuracy to detect precancerous lesions. Molecular-based screening approaches, such as human papillomavirus (HPV) DNA testing, which screens for oncogenic HPV infection, have shown high sensitivity for cervical precancer but can result in over-referral to colposcopy, a procedure to determine eligibility for treatment.The optimal screening test, age to begin screening, and frequency of screening for WLHIV remain uncertain.We evaluated several cervical cancer screening strategies in over 1,200 WLHIV in sub-Saharan Africa.We found that an HPV DNA test identified a greater number of women with precancer compared to visual inspection and cytology methods. However, there was a greater proportion of women without precancer who had a positive HPV DNA test, meaning a triage test using cytology or visual inspection was required to determine treatment eligibility.Simple user-applied modifications to the HPV-DNA-based test resulted in fewer women without precancer testing positive and fewer women needing triage.In settings with adequate infrastructure, cervical cytology was a useful triage test for HPV-positive women.Our data contribute to the evidence on choice of screening strategies for detection of cervical precancer among WLHIV in low- and middle-income settings.HPV DNA tests can play an important role in cervical cancer screening, especially in the era of universal antiretroviral therapy and where availability of self-sampling will facilitate screening participation.Prevention of cervical cancer should rank high as a public health priority in sub-Saharan Africa since WLHIV represent a group at very high risk of cervical precancer. In May 2018, the Director-General of the World Health Organization (WHO) announced a global call for action towards the elimination of invasive cervical cancer (ICC) as a public health problem, calling for more innovative technologies for detection of high-grade cervical intraepithelial neoplasia (CIN) grade 2 and higher (CIN2+) and better strategies to increase screening coverage and uptake  = 1/PPV) , and theWe evaluated the diagnostic accuracy of a restricted genotyping approach using results of the INNO-LiPA genotyping assay in the following combinations (positive for any genotype): HPV16; HPV16/18/45 (3 high-risk [HR] types), and HPV16/18/45/31/33/35/52/58 (8 HR types). Because of the low limit of detection of INNO-LiPA and to improve clinical relevancy, test positivity was defined as positivity for any of those genotypes among women who were also HC2 positive. We also evaluated the diagnostic accuracy of an HPV-based test targeting HR types previously reported to be most significantly associated with CIN3+ in the HARP cohort . RelativIn order to observe the performance of screening strategies in an already screened population, we evaluated the diagnostic accuracy of endline VIA/VILI, HPV DNA (careHPV), and cytology for cumulative CIN2+ detection at endline, excluding women who were treated for prevalent CIN2+ at baseline.10.17632/yd5ygw38vj.1.To evaluate the association of HIV-related factors with diagnostic accuracy of screening strategies, diagnostic accuracy for CIN2+/CIN3+ was evaluated separately among women on prolonged ART (>2 years), women on short-duration ART (≤2 years), and ART-naïve women at baseline . Diagnostic accuracy was also evaluated according to age at screening . The cumulative incidence of CIN2+ at endline was calculated among women who screened negative for each of the screening strategies at baseline . Analyses for diagnostic accuracy were conducted for discrete outcomes of CIN2+ and CIN3+. Data are presented separately for each country. Data were analysed using Stata (version 16) and according to the study statistical analysis plan . This arp < 0.001; p < 0.001).Of 1,395 women screened, 1,238 (89%) were enrolled in the HARP study , reflecting the 2:1 stratification ratio. Just over a third of women were taking ART for >2 years. The median CD4+ T cell count among women on ART for a prolonged duration (>2 years), women on ART for a short duration ART (≤2 years), and ART-naïve women was 478 (IQR 366–478), 394 (IQR 276–573), and 392 (IQR 310–591) cells/μl, respectively, in BF and 476 (IQR 372–626), 326 (IQR 207–453), and 440 (IQR 347–595) cells/μl, respectively, in SA. The corresponding values for HIV-1 plasma viral load were 40 (IQR 40–40), 40 (IQR 40–101), and 23,171  copies/ml, respectively, in BF and 109 (IQR 40–560), 141 (IQR 40–743), and 19,650  copies/ml, respectively, in SA.The median age of participants was 36 (IQR 32–42) years in BF and 34 (IQR 30–39) years in SA . About hp < 0.001). The prevalence of HR-HPV by HC2 was 41.8% (229/554) in BF and 59.7% (342/576) in SA  in BF and 79.3% (456/575) in SA (6) in SA .At baseline, positivity for each of the screening tests—VIA, VIA/VILI, ASCUS+, HSIL+, and HR-HPV DNA (HC2)—was 21.0%, 23.9%, 25.8%, 4.5%, and 41.8%, respectively, in BF, and 28.1%, 41.5%, 93.4%, 30.1%, and 59.7%, respectively, in SA .VIA had low sensitivity for CIN2+/CIN3+ , with specificity for ≤CIN1 of 78.7%  and specificity for ≤CIN1 . Increasing the threshold to ≥20 RLU resulted in fewer women screening positive  and increased the specificity to 71.4% (95% CI 68.4%–74.2%) but with loss in sensitivity . In BF, the number of colposcopy referrals was similar to current standard-of-care VIA/VILI , with a 2-fold increase in sensitivity for CIN2+  and CIN3+  but the lowest specificity  women using VIA or VIA/VILI had similarly low sensitivity for CIN2+ as using VIA or VIA/VILI as a screen test. In BF, although triage of HC2-positive (≥20 RLU) women with VIA/VILI had low sensitivity for CIN2+ , as was CIN3+ prevalence .In both countries, HR-HPV prevalence was higher among ART-naïve women (58.8%) and recent ART users (≤2 years’ duration: 60.6%) compared to prolonged ART users  compared to women on ART ≤2 years  or ART-naïve  for ≤CIN1 was higher in women on ART >2 years  compared to women on ART ≤2 years  or ART-naïve women , corresponding with lower HR-HPV prevalence in prolonged ART users . Consequp < 0.001; p < 0.001). There were no invasive cancer cases detected in either country at endline. Among 809 participants without CIN2+ at baseline, the incidence of CIN2+ over 16 months was 3.3% (95% CI 2.3%–4.8%) overall and was higher in SA .Of the 1,130 women evaluated at baseline, 1,042 (92.4%) were seen at the endline visit, at a median follow-up of 16.2 months (IQR 15.6–16.8), of whom 933 (89.5%) had histology data available at both time points  women in BF with CIN2+ detected at baseline who underwent management of their CIN2/3 lesions returned for the endline visit. In SA, 97 women with CIN2+ detected at baseline returned for the endline visit, and of these, 61 (63%) underwent management before the colposcopy/biopsy endline visit. Of the 36 participants who did not undergo treatment, 20 (55.6%) had CIN2/3 detected again at endline, and 16 (44.4%) had lower grade lesions (≤CIN1). Of the women who underwent management, the median time from colposcopy visit to management was similar in both countries .p-trend < 0.001). A test targeting HR-HPV type-specific persistence could detect 73.6%  cumulative CIN2+ cases at endline with a PPV of 16.9% (95% CI 12.3%–22.3%). The proportion of women without CIN2+ at endline and with HR-HPV type persistence was 22.6% .There were 903 women with matched histology and genotyping at both time points. Type-specific HR-HPV persistence was 20.7% (156/752) among women who were ≤CIN1 at both time points and 77.8% (21/27) among women with incident CIN2+. Among 87 women who received management for prevalent CIN2+, HR-HPV persistence was 37.0% (30/82) in those who remained ≤CIN1 and 66.7% (4/6) in women with CIN2+ redetected at endline . Among 3CIN2+ incidence at endline was 0.5% (95% CI 0.1–1.8) among women with a baseline negative HC2 (≥1 RLU) test or <LSIL on cytology, and 2.2% (95% CI 1.3%–3.7%) among women who were baseline VIA negative . Among HWe evaluated the diagnostic accuracy of screen and screen–triage approaches for CIN2+/CIN3+ in a large prospective cohort of WLHIV from 2 African countries with different HIV epidemics, different burdens of HPV infection and cervical cancer, and differing approaches to screening for cervical cancer. This allows the findings to be extended to a range of low- and middle-income settings. We found that an HPV-DNA-based test had high sensitivity but low specificity for CIN2+/CIN3+, but with simple modifications to increase the threshold for test positivity and with a restricted genotype approach resulted in higher specificity and correspondingly fewer referrals to colposcopy. Triage of HPV-positive women with VIA/VILI in BF and cytology (HSIL+) in SA resulted in a further reduction in referrals, with minimal impact on sensitivity for CIN3+, but not for CIN2+.HPV-based tests have high sensitivity for CIN2+/CIN3+ in both HIV-negative women and WLHIV, but specificity to distinguish CIN2+ is lower in WLHIV compared to HIV-negative women –34. HPV-We also found that the specificity of HC2 varied according to ART status, with the highest specificity observed in women taking ART for more than 2 years, corresponding with lower HR-HPV and CIN2+ prevalence. These findings are consistent with that reported in a cohort of WLHIV undergoing screening in Nairobi, Kenya , and JohAlternative approaches to the use of HPV tests could include repeat HPV DNA testing over time, which may distinguish HR-HPV persistent infection associated with CIN2+ from transient infections. We found in this study that 74% of WLHIV with CIN2+ detected at endline had type-specific persistence from baseline, compared with 23% of women without CIN2+ at endline. While such an approach may result in fewer women being unnecessarily treated or referred to colposcopy, the limitation is the potential for loss to follow-up of screen-positive women compared to a one-time HPV DNA test. On the other hand, repeat testing over a shorter interval  may be a feasible approach to integrate in routine HIV care, where WLHIV may be more frequently followed. Further data collection on the effectiveness and feasibility of such an approach is warranted.VIA is commonly used in LMICs, but we have shown it has low sensitivity for CIN2 lesions in WLHIV, consistent with other studies in Africa ,5,36, buCytology was the strategy with the best combination of sensitivity and specificity in SA, but only when the threshold for test positivity was increased to HSIL+. Similar high accuracy of cytology for CIN2+/CIN3+ has been reported in other studies in SA, which has an established cytology-based screening programme with quality control measures routinely implemented . StudiesThis study has several limitations. The study maximised the chances of obtaining histological results by basing the biopsy decision on positivity of any of 3 screening tests  or colposcopy , to which all participants were subjected . This approach and the threshold to trigger biopsy for histology are in excess of usual recommendations to minimise ascertainment bias. The number of post-biopsy adverse events was low; 6 (1.0%) women in BF and 4 (0.6%) in SA reported post-biopsy bleeding and/or abdominal pain. Women negative by all tests were considered to be at extremely low risk of CIN2+ since in particular HPV DNA and cytology have very high NPV for CIN2 diagnosis , and it HPV-based tests may be sufficient as a screening strategy in WLHIV if a restricted genotype approach is utilised and a higher threshold for test positivity is established. Molecular-based tests such as HPV tests have the added advantage of being automatable and less prone to training and interpretational errors than morphological tests such as VIA/VILI and cytology and can be performed using the same clinician-collected or self-collected sample, thereby simplifying sample collection, which may facilitate cervical cancer screening without the need for women to attend clinical services. Cytology remains optimal in settings with an existing cytology-based programme, such as SA. ART users with low or unknown nadir CD4+ cell count and ART-naïve women should be screened frequently, although the optimal screening intervals remain unclear. Although cervical cancer screening is not widely implemented in LMICs, integration of cervical cancer screening within HIV treatment services would ensure that women at high risk of developing cervical cancer precursor lesions are screened, and would lead to continuity in primary prevention, favouring early detection and management of HPV-related cervical lesions with minimal loss to follow-up . More loS1 Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S1 Text(PDF)Click here for additional data file. 1 Sep 2020Dear Dr Kelly, Thank you for submitting your manuscript entitled "Defining optimal cervical cancer screen and triage strategies for women living with HIV-1 in Sub-Saharan Africa: a diagnostic accuracy study" for consideration by PLOS Medicine.Your manuscript has now been evaluated by the PLOS Medicine editorial staff and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. 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Once your manuscript has passed all checks it will be sent out for review. plosmedicine@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Helen Howard, for Clare Stone PhD Acting Editor-in-ChiefPLOS Medicine plosmedicine.org 30 Sep 2020Dear Dr. Kelly,Thank you very much for submitting your manuscript "Defining optimal cervical cancer screen and triage strategies for women living with HIV-1 in Sub-Saharan Africa: a diagnostic accuracy study" (PMEDICINE-D-20-04079R1) for consideration at PLOS Medicine. Your paper was evaluated by a senior editor and discussed among all the editors here. It was also evaluated by three independent reviewers, including a statistical reviewer. 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Finally when completing the checklist for resubmission, please use section and paragraph numbers, rather than page numbers (because the latter are amended when the paper is reformatted for publication). -----------------------------------------------------------Comments from the reviewers:Reviewer #1: This is an interesting diagnostic accuracy study on cervical cancer screening and triage strategies for women living with HIV-1 in Sub-Saharan Africa. However, there are quite a few issues needing attention.1) The title of "Defining optimal cervical cancer screen and triage strategies..." is a bit misleading. This is basically an observational studies on the diagnostic accuracies of a range of cervical cancer tests on the two cohorts for BF and SA. To develop and validate a diagnostics test, needs to develop/establish a test in a training set and then validate the test in the independent/external validation set, the performance of the diagnostic test needs to be evaluated for both discrimination and calibration. So far, we didn't see any of the above in the paper, therefore it's mainly an observational study reporting on the test results of a range of diagnostic tests.2) As this is a diagnostic study, it's not clear what is the gold standard of all the tests. Also, it could be better structured and written in a way - primary outcome, secondary outcome, incremental test accuracies of test 1, 2 and 3. Now it's all over the place. What exactly is the research question? What's the rationale and link of involving two cohorts from BF and SA? It's like two separate studies. Also, what's the point of baseline and endline measurements? For the diagnostic studies, it is 'on the spot' test which is very different from prognostic test with follow-ups. Basically the follow-ups in the study didn't contribute to the evaluation of the tests. It would be good for authors to refer to the TRIPOD statement to the principles of diagnostic and prognostic studies.3) ROC curve is very important for the reporting of diagnostic tests. However, it's not mentioned at all in the statistics analysis section in methods. In Figure 2 and 3, there needs to be curves there not just the dots, and ROC values need to have 95% CIs. Also, the ROC values with 95% CI need to be presented in all the tables for test performance.4) In statistics analysis of the methods section, it says "We modelled the diagnostic accuracy of restricted genotyping approach using results of INNO-LiPA genotyping assay in the following combinations...", however, it's not clear what exactly the statistical model being used for this?5) Table 1. P-value is missing for the variable "HIV-1 PVL in ART users".-----------------------------------------------------------Reviewer #2: Thank you for this manuscript. This is a very important topic and a well written manuscript. In this time when the need for a specific cervical pre-cancer/cancer screening and prevention strategy for women living with HIV, such a research and manuscript adds to the accumulating evidence to inform policy making. I have very few comments and suggestions to help improve the manuscript:1. line 9: I suggest you make "cervical screening" to read cervical cancer screening. This will be clearer to the readers and also consistent with the rest of the manuscript2. Line 64-54: rephrase possible to read 'All participants were referred for colposcopy performed AT A median OF 12 weeks (interquartile range [IQR]: 8-15)...'3. tables 2-5: I understand you have tried to put all the required information on the tables but as they look now, they are difficult to read. Kindly see what can be done to improve the legibility. I think they are very important tables and so any efforts to make them more legible is important.4. line 425: it should read '.....test positivity IS established.....'-----------------------------------------------------------Reviewer #3: The authors tackle a key unanswered question for women living with HIV (WLHIV) globally. Cervical cancer has become the leading cause of death for WLHIV with access to ART in Africa, but achievable strategies to mitigate this burden have been illusive due to challenges of scale, prevalence of dysplasia, and pathology/cytology/PCR capacity. In this project, they prospectively enroll WLHIV in Burkina Faso and South Africa  to rigorously assess performance characteristics of visual, cytologic, and molecular approaches to screening. The core finding that molecular testing is superior  is a key piece of knowledge to move field/programs forward. However, as currently presented these findings are challenging for a general audience to gasps and understand importance. Revision for improved readability and focus could greatly improve impact.Suggestions:Major.1) Improve readability of abstract/manuscript for general medical audience- Abstract reads as though two different studies (BF and RSA), but could be improved by noting that evaluating in low and high/middle-income contexts with differing HIV and HPV prevalences for improved generalizability. Suggest considering merging findings as seems like molecular testing optimal strategy in both settings , for detecting cancers and reducing specialist-dependant services.2) More directly assess impact of weaknesses on findings/confidence. The key weaknesses include old study , unblinding of pathologists (how much bias would need to be present to modify findings?), use of careHPV , does different geography of HPV prevalence affect the conclusions, and can any intervention be effective if takes 8-14mo to treat dysplasia and 37% got no treatment?3) Age triage. Many programs considering threshold of 30yrs to improve specificity for HPV testing. Does this subgroup have different performance characteristics>Minor1) Unsure why some patients got VIA or VILI or both2) For an HIV audience, they would expect viral suppression as a categorical variable rather than continuous. And proportion with viral failure on ART (often >1000 copies)3) Introduction and elsewhere suggest relationship between HPV vaccination and HPVtesting, but not related4) Discussion long and lots of extrapolations re: CD4, ART timing beyond the data of this project. suggest focusing message.-----------------------------------------------------------Any attachments provided with reviews can be seen via the following link:[LINK] 21 Oct 2020AttachmentLetter of response_Reviewers_211020.docxSubmitted filename: Click here for additional data file. 9 Dec 2020Dear Dr. Kelly,Thank you very much for re-submitting your manuscript "Diagnostic accuracy of cervical cancer screening and screening-triage strategies among women living with HIV-1 in Africa" (PMEDICINE-D-20-04079R2) for review by PLOS Medicine.I have discussed the paper with my colleagues and the academic editor and it was also seen again by three reviewers. 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Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.Please also check the guidelines for revised papers at plosmedicine@plos.org) if you have any questions or concerns.We expect to receive your revised manuscript within 1 week. Please email us , which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.Please ensure that the paper adheres to the PLOS Data Availability Policy  and HC-II triage with VIA (2.1%) and lowest among HC-II screen-negative women (0.5%).”d. We had some difficulty locating the data in these statements for verification; please indicate which Table these can be found in:i. “Triage of HC-II positive women with VIA/VILI reduced the number of colposcopy referrals, but with loss in sensitivity for CIN2+ (58.1%) but not for CIN3+: (84.6%).”ii. “HC-II had higher sensitivity  but low specificity  resulting in almost twice as many referrals.”iii. “Compared to HC-II, triage of HC-II positive women with HSIL+ resulted in 40% reduction in colposcopy referrals but was associated with some loss in sensitivity.”e. Conclusions: Please start with “In this cohort study, …”f. Conclusions: Please mention WLHIV as well.6. Author summary: a. Please add “(WLHIV)” after first use.b. Please correct: “precancerous lesions cervical” to “precancerous cervical lesions” c. Please define/clarify “cytology” and “colposcopy” to a lay reader.d. 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N-(2-aminoethyl) pyridinium chloride salt as cationic groups, N, N-diethyl-1,3-benzenediamine as a coupling component, 2-amino-6-methoxybenzothiazole, 2-aminobenzothiazole or 3-amino-5-nitrobenzoisothiazole as diazo components. These dyes based on benzothiazole derivative chromophores not only showed beautiful color, including blue-green and fuchsia, but also had larger tinctorial strength with a high molar extinction coefficient, further reducing the dosage of dyes to achieve same color depth. Factors affecting the dyeability on fabrics, such as pH value, dyeing temperature and dye concentration were discussed. Excellent dyeing behavior, levelling properties and good fastness on wool/acrylic blended fabric were obtained. What’ more, excellent anti-ultraviolet and antibacterial properties were obtained for textiles with these dyes. The application of these dyes with large molar extinction coefficients presents a wide range of possibilities for the further development of cleaner production and eco-friendly dyeing, even functional textiles.Reactive dyes containing cationic groups have great potentiality as novel dyes, which can be applicable to one-bath dyeing of wool/acrylic blended fabrics. In this work, four novel heterocyclic reactive dyes containing cationic groups were designed by using m-aminophenyltrimethylammonium salt or  The application of blended fabrics greatly confers the complementary properties of fibers and reduces processing costs by combining the advantages of multiple fibers in textile goods. In recent years, the great market demand for wool/acrylic blended fabrics has remained a great impetus in knitwear because of its unique hand feeling, excellent wearing comfort, soft texture, and warmth retention ,2. GenerIn our previous work, it has proven promising to use reactive dyes containing cationic groups for the one-bath dyeing of wool/acrylic blended fabrics ,10,11,12Heterocyclic dyes are well-known compounds that have larger tinctorial strength and brighter color than azo dyes based on aniline-based derivative ,20,21, aS. aureus of dyed textiles were investigated.In the present paper, four reactive dyes containing cationic groups based on azo benzothiazole derivative shown in  as a diaN, N-diethylamino) acetanilide were received from Wanfeng Chemical Engineering Co., Ltd . All other reagents were obtained from Shanghai Chemical Reagent Co., Ltd. .Cyanuric chloride, 2-amino-6-methoxybenzothiazole and 2-aminobenzothiazole were supplied by the Tokyo Chemical Industry Co., Ltd.  and used without any further purification. Further, 3-amino-5-nitrobenzoisothiazole, 3-(2), acrylic (150 g/m2) and 50/50 wool/acrylic blended fabric (150 g/m2) were provided by Donghua University Textile Institute Training Center . The fabrics were pretreated with 2 g/L nonionic detergent at 60 ℃ for 30 min before being rinsed and dried.Wool .Ultraviolet-visible (UV-Vis) absorption spectra of these dyes, dissolved in dimethylformamide solution, were recorded with the U-3310 Spectrophotometer  using a quartz glass cell at the room temperature. The color strength (K/S) and colorimetric data of the dyed fabrics were determined by Datacolor SF 600X spectrophotometer . The topography of different fibers was observed at the optical microscope. A 3D microscope graph of fabrics was obtained by Super Depth of Field 3D Microscope VHX-6000 . Ultraviolet protection factor (UPF) and TN-(2-aminoethyl) pyridinium chloride salt and m-aminophenyltrimethylammonium salt were synthesized according to the reported researches [1H-NMR, and EA. The synthetic process and results can be found in These four novel dyes were provided by our research group. The cationic quaternary ammonium salts including searches ,36,37,38searches . MethanoAll dyeing of three kinds of fabrics were carried out using an infrared laboratory dyeing machine  using four heterocyclic reactive dyes with a liquor of 40:1 at different shade of 1~5% (o. w. f). These dyes were applied to wool/acrylic blended fabric using the one-bath dyeing method as described in max. The percentage of exhaustion (E%) was calculated using Equation (1):0 and A1 are the absorbance of the dye solution before and after the dyeing process, respectively.The absorbance of the dye solution before and after dyeing was carried out by UV-Vis spectrophotometry at λR is the reflectance of the dyed substrate at λmax, K is the absorption coefficient, and S is the scattering coefficient.In the spectrum visible region, 380–720 nm, the K/S values and color parameters  of the dyed fabric were measured using Datacolor SF 600X spectrophotometer. In order to obtain the correct K/S, five separate points of each tested dyed fabric were selected. The color yield of dyed fabrics was calculated using the Kubelka-Munk equation (Equation (2)).Considering that knitted fabrics were used for dyeing, fabric strength was obtained by bursting strength according to GB/T 19976–2005. The bursting strength of textiles was determined by the steel ball method. The maximum bursting strength of this sample was recorded at a constant rate of extension testing machine  when the sample was broken. Each kind of fabric was measured at least five times to obtain the mean bursting strength.Levelling test refers to the uniformity of the color of the fabric after dyeing. Five separate locations on the surface of the dyed fabric were chosen and assessed by Datacolor spectrophotometry ,40. ThenFastness testing for the dyed fabrics was performed according to international standards. Fastness to washing was assessed according to ISO 105-C03(2010). Fastness to the light was assessed using ISO 105-B02(2013). Fastness to rubbing was assessed following ISO 105-X12(2001).UVA% of fabrics were measured by Labsphere UV-2000F.Anti-ultraviolet testing for the dyed fabric was performed according to AATCC 183: 2014. The UPF and TStaphylococcus aureus (S. aureus) was engaged to perform the antibacterial testing of the dyed fabric according to AATCC100-2009, and the antibacterial rates were calculated by using Equation (4).max of them were 633.0 nm with greenish blue shade for D-1, 536.0, 535.0, and 542.0 nm with fuchsia shades for D-2, D-3, and D-4 in the visible region, respectively. It is clear to us that π-π* transition would be facilitated with the increase of the delocalized π-conjugated system [−1·cm−1, which was caused by the π-π* transition between the aromatic rings and the azo units.In order to investigate the relationship between spectral properties and the dye structures, the reactive dyes bearing different substituted groups were designed. The maximum absorption wavelengths of the four reactive dyes containing cationic groups with the same concentration (0.012 g/L) were measured in DMF solution. The strong absorption bands were observed in d system . The mol2 substituent group as the electron-withdrawing group, the maximum absorption wavelength of D-1, is found to demonstrate a remarkable bathochromic shift. The dye was obtained by replacing diazo component of D-1 structure with 2-amino-6-nitrobenzothiazole, which still had more bathochromic shift than D-2, but its λmax was shorter than that of D-1. It is known that the slight hypsochromic shift effect was usually caused by the introduction of electron-attracting groups into the “second leg” attached to the halotriazine [N-(2-aminoethyl) pyridinium as substituent. As observed in max was shifted towards a longer wavelength (bathochromism), which can be attributed to dye–solvent interactions by the dipolar effect [With the common coupling components, the color and depth of hues of these dyes were influenced by the electron effect of the substituent at the diazo components. Due to the introduction of -NOtriazine . D-3, m-r effect  and inter effect .onset at higher wavelengths according to Equation (5) [Further, the optical band gap tion (5) . This eqtion (5) . The optIn addition, in order to disclose the structure–spectra relation between these dyes, the DFT/B3LYP method combined with the 6–31 G (d) basis set was carried out with the Gaussian 09. The calculated energy gaps for chromophores in these dyes were 2.14, 3.11, and 2.84 eV respectively, which were in agreement with their UV-vis absorption spectra. A crude view of the processes can be provided by HOMO-LUMO plots . As can In the dyeing process, the dye-uptake is a major factor that should be taken into consideration. All kinds of fabrics were dyed by using an exhaust dyeing process. The types of cationic groups and chromophores presented in the structures of these dyes have a remarkable effect on dyeing performance . As illuN-(2-aminoethyl) pyridinium salt group.Optimized structures for the chromophore are shown in It is worthwhile to mention that the exhaustion of these dyes on acrylic fabric were higher than those on wool fabrics, while the K/S of dyed acrylic fabric was lower than those on wool fabrics. This implies that the color yield relates to differences of dye-uptake, dye distribution on fiber, the molecular structures of the dye, and the refractive index of fiber.The pH value is an important factor for different kinds of fabrics dyeing. With the exception of D-1, it can be seen from As displayed in It can be seen in An increase in the K/S values of dyed wool fabrics was observed with the increase of temperature, which attributed to the dense scale layers on the wool surface. The topography of wool fiber was observed by the optical microscope a. On theAdditionally, the K/S value tended to be smooth and even declined when the temperature exceeded 100 °C owing to the hydrolysis of the dye. As shown in Dyeing heat-curves of these dyes are described in As shown in The build-up property of three kinds of dyed fabrics is significant in terms of dye utilization and production cost , as showwool and (K/S)acrylic are the K/S values of dyed wool fabric and acrylic fabric, respectively.The union dyeing property (k) is an important index to determine the color match between dyed acrylic and wool fabric, which were calculated using Equation (4).The parameters  of the dyed fabrics at 1.0% (o. w. f) dye concentration are shown in Three fabrics were dyed with a liquor of 40:1 at 95 °C, 60 min, and 1.0% (o. w. f) dye concentration. The fabric strength of three fabrics was shown in The dyed wool/acrylic blended fabrics at 1.0% (o. w. f) dye concentration were measured by a Datacolor spectrophotometer to obtain the mean color differences (ΔE). As shown in N-(2-aminoethyl) pyridinium as cationic group had better light fastness than the other dyes, but they were grades 4–5 due to the benzothiazole structure.The color fastness properties of the dyed blended fabric with four dyes bearing an azo benzothiazole derivate at 1.0% (o. w. f) dye concentration were summarized in UVA% is less than 5% can be called the UV-protection products. Compared with untreated fabric, the UPF of dyed fabrics has been remarkably improved and the TUVA% of these four dyes was less than 0.5%. The data of UPF and TUVA% demonstrate that they exhibited excellent anti-ultraviolet properties. Further, results showed that the UV resistance of dyed wool/acrylic blended fabrics improved with increasing the dosage of four novel heterocyclic dyes owing to the increase of dye adsorption capacity.The effects of dyes on the anti-ultraviolet properties of dyed fabric were investigated and shown in Staphylococcus aureus was evaluated at 5.0% (o. w. f) dye concentration. As can been seen in According to the AATCC-100 antibacterial test method, the antibacterial rate of dyed wool/acrylic blended fabrics against Four novel reactive dyes containing cationic groups based on an azo benzothiazole derivative chromophores were designed and applied to wool/acrylic blended fabric by the one-union dyeing. These results demonstrate that these reactive dyes based on heterocyclic chromophores show beautiful color with high molar extinction coefficients. Moreover, these dyes exhibited excellent dyeing properties and fastness on wool/acrylic blended fabric. For wool/acrylic blended fabric dyed with D-1 and D-2, the optimum dyeing conditions were determined at pH 4, 95 °C for 60 min. In contrast, pH 6 is suitable for D-3 and D-4 dyes. Further, these dyes possessed excellent anti-ultraviolet properties and antibacterial activities. The present popularity of the reactive dyes containing cationic groups can be ascribed to their application of environmentally friendly functional finishing. This work will open the door for many dye chemists to improve the dye-uptake and color fastness and to enrich the applications of dyes on wool/acrylic textiles.


Escherichia coli (AIEC) pathotype has been associated with the aetiology of Crohn’s disease (CD). Scarce reports have shown the antimicrobial resistance (AMR) profiles of AIEC. Despite antibiotics not being recommended to treat CD, antimicrobial therapy could be useful in stratified patients, such as AIEC carriers. We examined the antimicrobial resistance profiles of AIEC strains to identify which therapies could be effective or confer a risk for such patients. Phenotypic resistance to 30 antimicrobials was tested according to CLSI standards. AIEC (n = 22) and non-pathogenic E. coli (non-AIEC) strains (n = 37) isolated from the gut mucosa of 31 CD patients and 18 controls were studied. De novo genome sequencing was carried out for 39 of the 59 strains, and AMR genes were searched using the DeepARG database in these genomes and 33 additional AIEC publicly available genomes. The strains isolated from CD and controls showed similar phenotypic AMR profiles. The genomic analysis did not reveal an increased prevalence of AMR genes. However, AIEC strains were more frequently resistant to β-lactams than non-AIEC strains (11 AIEC (50%) and 5 non-AIEC (22%) strains were resistant to at least one β-lactam; p < 0.042). Two AIEC strains were resistant to expanded-spectrum cephalosporins. One strain carried a plasmid-mediated AmpC β-lactamase (CMY-69), and the other presented mutations in the promotor of the intrinsic chromosomal AmpC related to the hyperproduction of this enzyme. The rest of the strains were resistant to β-lactams not including expanded-spectrum cephalosporins. The majority carried TEM-related β-lactamases. Genomic analysis including external AIEC revealed that the gene sul1 encoding for sulphonamide resistance was more frequent in AIEC strains than non-AIEC strains . AMR in AIEC is a matter of concern regarding the putative implication of the pathotype in CD. The high proportion of AIEC resistant to β-lactams warrants caution about the risk there may be in the use of these antimicrobials in AIEC-colonized CD patients.The adherent invasive  Escherichia coli (AIEC) pathotype was described in 1999 when it was discovered in association with Crohn’s disease, a chronic inflammatory bowel disease [The adherent invasive  disease ,6,7,8,9. disease ,11,12.E. coli (ExPEC) [The AIEC pathotype is defined by its ability to adhere to and invade intestinal epithelial cells, as well as its capacity to survive and replicate within macrophages while inducing the secretion of tumour necrosis factor α (TNF-α) . AIEC st (ExPEC) . The vir (ExPEC) .Since AIEC and other pathogens have been repeatedly associated with the aetiology of Crohn’s disease, antibiotics could be a logical approach to therapy, especially those that are effective intracellularly. The therapies that are currently applied to inflammatory bowel disease are mainly directed at the suppression of the immune system. They involve anti-inflammatory drugs , corticosteroids , immunosuppressant drugs (azathioprine and mercaptopurine), biological therapy (TNF-α inhibitors), faecal transplantation or nutritional interventions, and surgery ,16. A syE. coli isolates from Crohn’s disease patients and six from controls. Taking into account the clinical relevance of AIEC in Crohn’s disease, it is valuable to have a better knowledge of the antimicrobial resistance in further AIEC collections and to gain insights about the molecular mechanisms of resistance.Few studies have reported on the antimicrobial resistance of AIEC. Dogan et al.  studied E. coli strains that were isolated from Crohn’s disease patients and control subjects to 30 antimicrobials, which could be useful in the design of future clinical trials for the disease. We have also determined the putative mechanisms of resistance of the considered strains in vitro and in silico and compared them with the antimicrobial resistance genes present in other AIEC genomes in public databases.In this work, we examined the antimicrobial resistance profiles of 49 p = 0.042). In particular, 50% of AIEC strains were resistant to penicillins such as ampicillin and ticarcillin, and between 18.2% and 31.8% of them were also resistant to penicillins combined with inhibitors of β-lactamases. Moreover, 18.2% of AIEC strains were resistant to first- and second-generation cephalosporins, and about 10% were resistant to expanded-spectrum cephalosporins. However, none of the non-AIEC strains were resistant to those cephalosporins. Moreover, a higher frequency of strains resistant to cefoxitin was also detected in the AIEC collection, although the result did not reach statistical significance.Detailed antimicrobial resistance profiles of the strains are presented in p = 0.134 and p = 0.136, respectively).The proportion of strains resistant to other antimicrobials such as tetracyclines, aminoglycosides, and quinolones was similar between AIEC and non-AIEC strains . Moreovep = 0.029).No differences were found in the frequency of resistances between strains isolated from Crohn’s disease patients and control subjects   or in thTwo patterns of antimicrobial resistance to β-lactams were found in the AIEC collection . Eight AIn particular, AIEC08 and LF82 exhibited resistance to ampicillin, ampicillin/clavulanic acid, ticarcillin, ticarcillin/clavulanic acid, cefazolin, cefoxitin, cefuroxime, cefotaxime, ceftazidime, cefpodoxime, cefovecin, and ceftiofur . This anampC region of E. coli K12, strain LF82 contains mutations at positions −73 (C to T), −32 (T to A), −28 (G to A), +17 (C to T), and the novel +23 (G to A). The T to A transversion at position −32 creates the consensus TTGTCA −35 box responsible for an increased transcription of ampC  are associated with the phylogenetic origin of the strains. The sequence of the chromosomal ampC promotor in AIEC02 was identical to that of strain E. coli K12 , which raises questions about their implication in coli K12 . Thus, nsul1 was more prevalent in AIEC strains .We studied the genomic resistance profiles of internal and external strain collections to describe additional resistance genes apart from those related to β-lactams and infer the antimicrobial resistance in additional AIEC strains. The precise antimicrobial resistance (AMR) gene carriage of each strain is shown in IntI1) gene was found in 16 out of the 18 sul1-positive strains, suggesting that the resistance gene was present in class 1 integrons. The two other strains, AIEC25 and B2_33-1-TI5, showed partial matches with very high identity to intI1 but corresponded only to 40–50% of the length of the intI1 gene. Fortunately, extended-spectrum β-lactamases were not present in any other strain apart from the Spanish AIEC08 strain. However, TEM-148 was also present in the American strains MS124-1, MS85-1, MS107-1, MS79-10, and MS57-2 and the Australian strains B2-33-1-TI5, B2-52-1-TI13, B2-61-1-TI1, and B2-CD-62-LN.The class 1 integron-integrase (E. coli isolated from controls (17.6% vs. 0%), and two genes related to aminoglycoside resistance (strA and strB) were more frequently found in E. coli strains isolated from Crohn’s disease patients .A similar AMR gene distribution was also observed between strains isolated from Crohn’s disease patients and those isolated from control subjects B. As an AIEC colonizes the intestine of 25–75% of Crohn’s disease patients ,7,8,13, E. coli strains of the clone ST131 O25:H4/H30-Rx with resistance to extended-spectrum β-lactams that had AIEC virulence traits. Further studies are needed to confirm whether resistance to β-lactams is a frequent trait in other AIEC collections.The most relevant result of this work is that AIEC strains were more frequently resistant to β-lactams than non-AIEC strains isolated from the intestine of Crohn’s disease and control subjects. Few studies on the antimicrobial resistance in AIEC have been published. Among them, Dogan et al.  showed nampC promotor that could be related to increased AmpC expression. Genomic analysis of AMR gene carriage in other publicly available AIEC genomes showed a similar pattern of genetic mechanisms of resistance. These strains had at least a chromosomic ampC β-lactamase, and nine of them also harboured TEM-148, which corresponded to 30% of the strains studied, which is a very similar proportion to that in our strain collection (33%). Fortunately, none of the external AIEC strains had genes encoding for extended-spectrum β-lactamases. However, a recent work has demonstrated the presence of CTX-M15 and OXA-1 extended-spectrum β-lactamases in four AIEC strains isolated form healthy subjects [Mechanisms of resistance against β-lactams included non-extended-spectrum β-lactamases TEM-148 and TEM-1 and the extended-spectrum β-lactamase CMY-69, as well as mutations in the subjects .sul1 gene in AIEC strains in comparison to non-AIEC strains. Sul1 is a sulphonamide-resistant dihydropteroate synthase that is normally found linked to other resistance genes located in Class 1 integrons. The intI1 gene was found in the genomes of the majority of sul1-positive AIEC strains, which confirmed the presence of Class 1 integrons in these strains. Only two sul1+ strains were intI1–, but partial sequences with high identity were found. Thus, poor genome quality could have hampered the identification of the intI1 gene in these genomes. Alternatively, the sul1 gene could be present in the chromosome or other genetic elements that are different from class 1 integrons in these strains [sfaS and siderophore receptor genes fyuA (ferric yersiniabactin receptor gene) and iutA (ferric aerobactin receptor gene) [Genomic analysis of AMR genes also revealed an increased frequency of the  strains . Class 1or gene) .E. coli from Crohn’s disease patients than those from healthy subjects. Particularly, there was a higher frequency of strains resistant to ciprofloxacin, rifaximin/rifampin, and trimethoprim/sulfamethoxazole.A similar proportion of resistant phenotypes was found for all the antimicrobials tested between strains isolated from Crohn’s disease patients and control subjects, with the exception of rifampin. In contrast to our observations, Dogan et al.  found a E. coli strains isolated from either Crohn’s disease patients or controls. Despite a breakpoint for rifampin not being available for E. coli, we found a higher proportion of strains with MIC values >2 mg/L in Crohn’s disease E. coli strains than in control E. coli strains. A non-absorbable variant of this antibiotic (rifaximin) has been commonly used in the management of inflammatory bowel disease [E. coli strains could be the selection of resistant strains due to the previous consumption of rifampicins by such patients, but unfortunately, we do not have access to this information. In a previous study [E. coli strains isolated from ileal Crohn’s disease patients and in none of the E. coli isolated from healthy subjects, and strongly correlated with prior exposure of patients to rifaximin (p < 0.01).In line with our results, Subramanian et al.  and Elio disease . A possius study , rifaximE. coli strains isolated from Crohn’s disease patients and controls, and some of them have the AIEC phenotype. We found a low frequency of ciprofloxacin-resistant strains (around 4%), so this antimicrobial could be considered to treat patients overcolonized by E. coli or specifically AIEC.Antimicrobials used for the management of Crohn’s disease have mainly been anti-mycobacterials, rifampins, metronidazole, clarithromycin, and ciprofloxacin . DespiteClinical trials in Crohn’s disease patients have demonstrated that this fluoroquinolone can be useful to treat Crohn’s disease fistulas . MoreoveE. coli strains were isolated to test this hypothesis.Clinical management of Crohn’s disease does not generally include β-lactam treatment, but this group of antibiotics is frequently used to treat many human and animal infections. However, it has been reported that the repeated use of cephalosporins and extended-spectrum penicillins during childhood was associated with increased risk of Crohn’s disease in a Finnish cohort . We hypoAn elegant study based on animal models demonstrated that the use of streptomycin, vancomycin, and gentamicin can promote the initial infection of AIEC and worsen infection in previously colonized hosts . The exaThis study provides new evidence of antimicrobial resistance in AIEC and non-AIEC strains isolated from the intestinal mucosa of Crohn’s disease and control patients and, to our knowledge, this is the first study in which a genome-based analysis has been performed to identify genetic mechanisms of antibiotic resistance in AIEC. Antimicrobial resistance in AIEC is a matter of concern regarding the putative implication of the pathotype in Crohn’s disease. Before prescribing any antimicrobial therapy for a Crohn’s disease patient, we believe that the identification of AIEC carriers and the characterization of the antimicrobial resistance profiles of AIEC from these AIEC carriers could be very relevant to preventing bad clinical outcomes for patients. Moreover, considering the impact that antimicrobial treatments have on the gut microbiota composition, even promoting the colonization and expansion of AIEC in some cases, alternative precision antimicrobial therapeutics should be further explored, such as FimH  antagonists , colicinE. coli strains were isolated from the intestinal mucosa of Crohn’s disease patients  and control subjects  [E. coli (non-AIEC). The study was approved by the ethics committee of clinical investigation of the Hospital Josep Trueta of Girona. Patients did not receive antibiotics for two months prior to colonoscopy. This strain collection is referred as “internal strains” in this study. The main characteristics of the strains are detailed in In a previous work, a collection of 49 ®2 system .We selected the most used antimicrobial agents in clinical and veterinary practice, including a large variety of antimicrobial categories. The isolates were tested for resistance to ampicillin, amoxicillin/clavulanic acid at a 2:1 ratio, piperacillin/tazobactam (tazobactam at a fixed concentration of 4 mg/L), cefuroxime, cefoxitin, cefotaxime, ceftazidime, cefepime, ertapenem, imipenem, amikacin, gentamicin, nalidixic acid, ciprofloxacin, tigecycline, and trimethoprim/sulfamethoxazole. The tests were conducted using the VitekResistance to ticarcillin, ticarcillin/clavulanic acid (fixed concentration of 2 mg/L of clavulanic acid), cefazolin, cefovecin, ceftiofur, cefpodoxime, enrofloxacin, marbofloxacin, doxycycline, chloramphenicol, and rifampin was further tested using a COMPAN1F Sensititre standard susceptibility plate . Isolates were also tested for resistance to tetracycline, kanamycin, and streptomycin by a macrodilution test according to the standards of the Clinical and Laboratory Standards Institute (CLSI) .The MICs were interpreted according to the CLSI guidelines . For theE. coli. To test this possibility and test for the presence of an ESBL, a phenotypic disk diffusion assay on Mueller Hinton agar plates was performed with or without 250 mg/L of cloxacillin and discs of cefotaxime, ceftazidime, and cefepime with and without clavulanic acid in all three cases. Cloxacillin has an inhibitory effect on AmpC, so the test was considered positive if the diameters of the inhibition zones were ≥5 mm in the plates with cloxacillin [Two strains presented an antimicrobial resistance profile that suggested the presence of either a plasmid-mediated AmpC-type β-lactamase or hyperproduction of the intrinsic chromosomal AmpC of xacillin . Moleculxacillin .Thirty-nine strains (18 AIEC and 21 non-AIEC) out of the 49 strains analysed phenotypically for antimicrobial susceptibility were sequenced. Genome assemblies were generated starting from Illumina MiSeq  raw sequencing reads (2 × 300 bp). Overlapping read pairs were stitched using FLASH v1.2.6 software with the following options: —max-mismatch-density = 0.25 —min-overlap = 10 —max-overlap = 300. De novo assembly of the stitched reads was performed with CLC-Assembly Cell 4.2.2  with default parameters.E. coli genomes were analysed, including the genomes of the 18 AIEC and the 21 non-AIEC strains that were internally sequenced, as well as 33 additional AIEC genomes that were retrieved from public databases  [In total, 72 strains) ,43,44,45strains)  in singlstrains)  to identampC genes were analysed separately. For the three plasmids, no ampC gene sequence was identified. In total, the nucleotide sequences of ampC genes for 72 samples, including 200 bp upstream of the starting codon, were retained and aligned using Muscle v3.8.31 (https://www.drive5.com/muscle/) [https://biopython.org/) [E. coli strain, which was set as the zero coordinate. The lists of antibiotic resistance genes and nucleotide variations on the ampC genes for each sample were compiled into two separate tables using Pandas v0.23.3 (https://pandas.pydata.org/).All the genes annotated as members of the β-lactamase class of resistance were isolated, and the ones corresponding to the muscle/)  with defon.org/)  was usedintI1 gene sequence from a representative E. coli strain (O104:H4) was downloaded from the Ensembl Bacteria database . The intI1 gene sequence was searched in the predicted genes for the 72 strains and the 3 plasmids using BlastN  with the following parameters: -word_size 11 -perc_identity 95. The alignment results were further filtered to retain only results where at least 90% of the intI1 representative gene sequence was aligned on one of the genes for a particular strain. The analysis pipelines and workflows were executed using Nextflow  [For the Class I integrase gene search, the , Spain) .2 or Fisher exact tests . Quantitative data were compared by the Mann–Whitney U test. Statistical analyses were performed using SPSS IBM Statistics 25 15.0 software .Differences in the frequency of resistant strains between groups (AIEC vs. non-AIEC and Crohn’s disease vs. controls) were tested with Pearson X


Estimate the incidence of herpes zoster (HZ), its complications and healthcare utilization rates in adults (≥ 18-years-old) with a wide range of immunocompromised (IC) conditions compared to IC-free cohort.A population-based retrospective study using the Valencia healthcare Integrated Databases (VID) (2009–2014). HZ and IC were defined using ICD-9 codes in primary care (PC) and hospitalization registers. Incidence rates (IR), risk of HZ, HZ-recurrence, HZ-complications and healthcare utilization rates were estimated in the IC-cohort compared to IC-free.The study population consisted of 4,382,590 subjects, of which 578,873 were IC (13%). IR (in 1000 persons-year) of HZ overall, in IC and in IC-free cohort was 5.02, 9.15 and 4.65, respectively. IR of HZ increased with age in both cohorts and it was higher for all IC conditions studied, reaching up to twelvefold in subjects with stem cell transplantation. IC subjects had 51% higher risk of developing HZ, 25% higher HZ-recurrence and the risk of HZ-complications was 2.37 times higher than in IC-free. HZ-related healthcare utilization was higher in the IC-cohort than in IC-free .Patients suffering from all the IC conditions studied are at higher risk of developing HZ, HZ-recurrence and post-herpetic complications, which implies a substantial morbidity and a high consumption of resources. These results should be considered for vaccine policy implementation. One in three people aged between 50 and 90 years will develop herpes zoster (HZ) and approximately one in ten will develop postherpetic neuralgia  . A decreBoth, HZ and PHN result in reduced quality of life as well as individual and societal healthcare costs –8. Its tPrevious studies on HZ and PHN epidemiology using Real World Data have been developed in order to identify highly susceptible populations such as patients with diabetes, chronic obstructive pulmonary disease (COPD) or heart failure (HF) among others , 16–18. Mareque et al., in Spain there is a lack of these types of studies and its heterogeneity and limited populations make it difficult to develop the definition of priority groups for the implementation of a future non-routine HZ vaccination program [The estimation of the full burden of HZ disease including HZ incidence and recurrence rates, its complications and the evaluation of HZ risk for specific IC subpopulations constitutes a critical step to make an estimation of future impact of the RZV in these risk groups. So far, only a few studies assessed HZ incidence among patients with different IC conditions including populations large enough to allow comparison among the different conditions , 26, 27. program .The Valencia Region of Spain has a population of around 5 million inhabitants, over 96% of which are insured by the Regional Health System (RHS) . The RegThis is a population-based retrospective cohort study using real-world data from the VID, including a population ≥ 18 years old living in the Valencia Region between 2009 and 2014. The inclusion date was defined as 1st January 2009 for those subjects older than 18 years continuously registered in the RHS for at least 12 months before this date, or first date after 1st January 2009 when the subject was continuously registered in the RHS for at least 12 months before this date and was 18 years old. The date of end of follow-up was defined as end of the study period (31st December 2014) or the date of exit of the RHS (including death), whichever comes sooner.We used the following registries from VID : 1) the IC-cohort consisted of all eligible individuals with an IC condition-related code registered in SIA or MBDS as follows: HSCT, solid organ transplantation (SOT), haematological neoplasia (HN), solid organ neoplasia (SON), neoplasias overall, HIV, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), IBD, psoriasis, multiple sclerosis (MS), autoimmune thyroiditis (AT) and immunodeficiency disorders and autoimmune diseases overall (autoimmune) (See supplementary Table 1 for ICD-9-MC codes) [The C codes) , 34. SubIC-free cohort consisted of subjects with no IC condition-related code detected during the study period. The start of follow-up for the IC-free cohort was the date of inclusion in the study. The IC-free status of an individual changed during the study period if an ICD code for an IC-related condition appeared in MBDS or SIA databases. In the case of PC or hospitalization IC diagnoses, the beginning of IC status was defined as the date in which IC disease was diagnosed and the subject was considered IC from this date until the end of the follow up period (end of the study period or exit from SIP including death).The HZ-incident cases were considered as the first appearance of a HZ-related ICD-code (053.x)  in eitheHZ-recurrent cases were considered when new HZ-related ICD-code appeared at least 6 months after a previous HZ encounter in the same subject. HZ case identification using databases and ICD-codes have shown elevated positive predictive value  .HZ-complications were defined as any HZ-related hospitalization with one of the following ICD-codes at discharge in MBDS: central nervous system (CNS) complications (053.0 and 053.1x) which include PHN , ophthalmic complications (053.2x) and other complications (053.7x and 053.8x).Socio-demographic characteristics ) of the study population and IC condition type (for the IC cohort), were summarized using descriptive tables including proportions and frequencies.HZ incidence (number of cases per 1000 persons-year) and recurrence (number of cases per 100 persons-year) rates were estimated for IC population, globally and stratified by gender, age groups: 18–29, 30–39, 40–49, 50–59, 60–69, 70–79 y ≥ 80 years, calendar year and type of IC condition. The respective 95% confidence intervals (CI) were calculated by the exact method of Poisson.The risk of HZ, HZ recurrence and HZ complications in the IC-cohort subjects respect to IC-free cohort were estimated by a Poisson  regression adjusted by age, gender, calendar year, comorbidities , and health department.Healthcare resource utilization among IC and IC-free cohorts was estimated as the number of HZ-related PC visits, number and length of HZ-related hospitalizations, number and duration of sick leaves due to HZ and prescriptions and dispensations of antivirals  during the six months following the HZ diagnosis. We performed different statistical General Linear Models (GLM) to compare the populations with and without IC conditions . This cohort included 578,873 (13% of the study population) individuals with IC . Among the IC conditions, neoplasia (7%) was the most frequent, including SON (5.9%) and HN (1.3%). They were followed by psoriasis (3.3%) and RA (0.9%). Diabetes was registered in 11.2% of the population, COPD in 5.1%, CKD in 3.4% and HF in 3.2%. Some conditions were more frequent among men such as HIV (71.4%) or COPD (66.2%) and some other among women including SLE (84.2%), MS (68.9%) or RA (68.2%). Sample size overall and by IC condition in the IC-cohort and IC-free, age distribution and demographic characteristics are listed in Table Incidence rate (IR) (in 1000 persons-year) of HZ  was 5.02 in population overall, 9.15 in IC cohort and 4.65 in IC-free cohort. IR of HZ increased with age, in IC-free cohort the lowest IR was 2.26 in 18–29 age group, and the highest IR was 9.54 in ≥80 age group. In IC cohort the lowest IR was 3.61 in 18–29 age group and the highest was 12.93 in 70–79 age group . The overall IR of HZ was higher in women than in men in both IC (9.74 vs. 8.48 cases/1000 persons-year) and IC-free cohorts (5.43 vs. 3.83 cases/1000 persons-year)  of HZ in the studied IC conditions ranged from 6.13 for psoriasis to 56.07 for HSCT Table . IR for The adjusted risk of HZ increased by 51% among people with IC conditions with respect to people IC-free . It was 33% higher among females than that of males  and it increased with age  of HZ related complications was 3.63 in population overall, 14.36 in the IC cohort and 2.64 in the IC-free cohort  of HZ were 1.66, 2.28 and 1.56 in the population overall, the IC and the IC-free cohorts, respectively. The highest recurrence rate was found in people with HSCT (5.5/100 persons-year), followed by people with HN (3.35/100 persons-year) and HIV (3.2/100 persons-year)   population-based study to date investigating the incidence of HZ, its complications and healthcare resources utilization in ≥18-years-old adults in Spain with a wide variety of IC conditions . Our stuThe observed IR of HZ (5.02/1000 persons-year) for the overall study population  is aligned with previous publications , 36. DesIn our study, the IC-cohort represented around 13% of the study population. This percentage is difficult to compare among previous studies from different countries in which the values fluctuate from 1.86 in Japan  to 16.8 Considering all HZ-related complications, we showed that IC patients have more than twice the risk of hospitalization by a HZ complication than IC-free people. 70% of the HZ-related complications were associated to the CNS, with PHN as the most common. In our previous studies we described that 15.7% of the total HZ cases developed in people above 50 years old resulted in PHN lasting for at least three months after the acute phase of a HZ , 17. NotMost of the publications agree that the risk of developing an HZ rises significantly with age, most likely caused by the decreasing cell-mediated immune response to the VZV due to the immunosenescence , 37. ThiThe recurrence rate of HZ ranged from < 1.5 to 6.8% among different studies . ResultsIn addition to age and comorbidities, female gender has also shown an increased risk of developing HZ and suffering recurrences and PHN. This result is also in concordance with previous publications –40. It hThe greatest strength of our study is, without a doubt, the large study population and the VID, a network of electronic health databases where all the information on each subject  can be linked at the patient level. However, some of its limitations are worth mentioning. Firstly, HZ-related hospitalization rates found are higher than other previously published. It probably represents an overestimation as we considered hospitalizations with HZ diagnoses in any diagnostic position and HZ could represent a secondary diagnosis. Secondly, the proportion of IC population increased during the study period. One of the reasons could be related to IC-cohort case definition, since once a diagnosis of a chronic IC condition was registered the patient remained in the IC-cohort group until the end of the follow-up period. It should also be considered that the individuals with none of the selected IC conditions diagnosed could be not necessarily immunocompetent. On the other hand, severity grade of the studied IC conditions and IC-related therapies have not being considered in this study which may impact the HZ IR and its complications. This lack opens the way for new research in this regard looking for associations between the severity or therapy and time to HZ episode.As we previously have shown for some other underlying conditions such as Diabetes, COPD, or HF , 17, theIn this study we estimate the burden of HZ disease in people with IC conditions in a Spanish Region of 5 million inhabitants. All the IC conditions studied were above the estimated overall IR in the IC-free cohort and the IR of HZ complications was about 5 times as high in the IC as in the IC-free cohorts. In conclusion, the selected IC conditions increase the risk and severity of HZ episodes and result in higher healthcare resources utilization. This first step should be followed by cost effectiveness studies that help us establishing the value of vaccinating these groups of patients when the new prevention strategies become approved in those risk groups.Additional file 1: Supplementary Table 1. CIE-9-MC codes for immunocompromised.Additional file 2: Supplementary Table 2. Generalized lineal models.


Orexin-containing neurons exist in the lateral hypothalamic region, sending their projections toward mesolimbic regions such as the Ventral Tegmental Area (VTA).In the current study, a Reinstatement model is used to examine the effects of intra-VTA administration of SB334867 as an Orexin-1 Receptor (OX1R) antagonist on drug priming- and Forced Swim Stress (FSS)-induced reinstatement of morphine. Eighty-eight male adult albino Wistar rats, weighing 200–280 g, were bilaterally implanted by cannulas into the VTA. We induced the Conditioned Place Preference (CPP) by Subcutaneous (SC) injection of morphine (5 mg/kg) daily in three days. Then, the CPP score was calculated. After a 24-h “off” period following achievement of extinction criterion, the rats were tested for drug priming-induced reinstatement by a priming dose of morphine  and for FSS-induced reinstatement 10 min after FSS. In the next experiments, the animals received different doses of intra-VTA administration of SB334867  and bilaterally were subsequently tested for FSS- and morphine priming-induced reinstatement.Our findings indicated that the FSS could induce the reinstatement of seeking behaviors. Furthermore, intra-VTA administration of OX1R antagonists suppressed FSS- and drug priming-induced reinstatement dose-dependently.It is concluded that FSS and drug priming-induced reinstatement might be mediated, at least in part, by stimulation of orexin receptors in the VTA. Forced swim stress (FSS) induced the reinstatement of morphine seeking in rats.Orexin-1 receptor blockade in the intra-ventral tegmental area reduced morphine-induced reinstatement.Orexin-1 receptor blockade in the intra-ventral tegmental area reduced FSS-induced reinstatement.We designed this study to investigate effects of intra-VTA  administration of SB334867 as an orexin-1 receptor (OX1R) antagonist on drug priming- and forced swim stress (FSS)-induced reinstatement of morphine. The conditioned place preference (CPP) was induced by injecting morphine and the reinstatement by the administration of effective priming dose of morphine. The extinguished rats received an intra-VTA injection of SB334867 before effective priming dose injection of morphine. In others, the extinguished rats were given intra-VTA administration of SB334867 bilaterally and were subsequently tested for FSS- and morphine priming-induced reinstatement. Our results indicated that intra-VTA administration of SB334867 could inhibit morphine priming - and FSS-induced reinstatement of extinguished morphine seeking in the rats. It seems that OX1R in the VTA may be involved in the reward system and could play an important role in the effect of stress on reinstatement of morphine-seeking behaviors in this area.1.Drug addiction can be conceptualized as a recurrent disorder characterized by the high rate of relapse to drug taking following long periods of abstinence . ExposurThe reinstatement Conditioned Place Preference (CPP) model has been widely used in many laboratories as an animal model of relapse to drug seeking  that indSeveral orexin projections associated with behavioral responses of drug abuse include the Nucleus Accumbens (NAc) and Ventral Tegmental Area (VTA) . All addMultiple lines of evidence have shown that orexin transmission play a key role in expression, extinction, and reinstatement of drug .It is noIn the present study, we tested whether the infusion of the OX1R antagonist, SB334867, into VTA modifies the relapse of morphine-seeking elicited by FSS and morphine priming previously associated with morphine availability.2.2.1.The experiments were performed on 88 adult male albino Wistar rats  which were housed on a 12:12 h light/dark cycle at a temperature controlled room with free access to food and water. All methods used complied with guidelines for Care and Use of Laboratory Animals  and were approved by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran.2.2.The rats were mounted on a stereotaxic apparatus  under ketamine 10% (100 mg/kg) and xylazine 2% (10 mg/kg) anesthesia. Then, using a stereotaxic apparatus, two guide cannulas were implanted 1 mm above the VTA . These coordinates are in accordance with the rat brain atlas of Paxinos and Watson . The gui2.3The drugs used in the present study were morphine sulfate  and SB334867 as a selective OX1R antagonist . Morphine sulfate was dissolved in saline and SB334867 was dissolved in dimethyl sulfoxide . The control animals received either saline or 12% DMSO or both as the vehicle.2.4.Microinjections were performed by attaching an injection needle (30-gauge injector cannula) to a Polyethylene tubing (PE-20). The free end of the tubing was attached to a 1-μL Hamilton syringe. An appropriate amount of injection was drawn (VTA: 0.3 μL/rat) into the tubing, and was infused over 60 second while the animal roamed freely.2.5.2.5.1.The apparatus contains three-compartment conditioning boxes. The third one of which is considered as a start box, varying in size (30×15×40 cm) and usually serves as a connection between the other two equal-sized compartments (30×30×40 cm) and is separated by a removable Plexiglas wall. Conditioning took place in one of two compartments, which differed in pattern and texture. One compartment had white backgrounds with black vertical stripes and a smooth floor. Another compartment had black horizontal stripes with a rough floor. Before the conditioning session, the animals displayed no baseline preference for either of these two compartments.2.5.2.The Conditioned Place Preference (CPP) involves three phases: pre-conditioning, conditioning, and post-conditioning.Pre-conditioning phase: In the pre-conditioning procedure (day 1), through the removal of the guillotine door, the animal was allowed to access all compartments freely. It was conducted to determine the baseline chamber preference and consisted of one 10-minute trial for each rat. Each animal displacement was recorded.Conditioning phase: One day after the pre-conditioning session, the conditioning phase started consisting of six 30-min sessions  in a 3-day schedule (the second day to the fourth day). These sessions were carried out twice each day, and the animals were restricted for 30 min after the first injection to one side of the two-sided compartment, returned to their home cages for 6 h, and then subjected to another 30-min conditioning trial. On each day, separate groups of animals received a conditioning session, one with drug and another with saline. The treatment compartment and order of presentation of drug/saline were counterbalanced for either group. Based on our recent studies , 5 mg/kgPost-conditioning phase: Each rat experienced a single CPP assessment session 24 h after the last conditioning session and done in a drug-free state. In this session, the rats were tested only once, having access to both compartments. The time spent and the distance traveled in each compartment for each rat during a 10-min period were recorded by a 3CCD camera  and analyzed using the EthoVision software , a video tracking system for automation of behavioral experiments . The conditioning score, as a preference index, was calculated as the time spent in the drug-paired compartment minus the time spent in the saline-paired compartment . The tot2.6.After the conditioning procedure, the animals were located in the CPP apparatus (on days 6–13) in a drug-free state (extinction period) and CPP scores were calculated every day . Extinct2.7.During this procedure, the rats were exposed on a 6-min forced to swim in a plastic cylindrical tank measuring 50 cm length ×30 cm width, that was filled with clean water (23°C–27°C) with a height of 30 cm . After t2.8.2.8.1.Five groups of rats were used in this set of tests. There were two control groups; in non-FSS (NFSs) control group, the vehicle was infused into the VTA (0.3 μL 12% DMSO per side), and in another control group, the vehicle was infused into the VTA (0.3 μL 12% DMSO per side) 5 min before FSS. In the other three groups of rats, different bilateral doses of SB334867  were administered into the VTA, 5 min before FSS. Ten minutes after FSS, all three groups of rats were located into the CPP boxes for the Reinstatement-test and permitted to move freely for 10 min. The CPP score and the distance traveled were documented .2.8.2.In this set of experiments, five groups of animals were used. In one control group, intra-VTA vehicle (0.3 μL 12% DMSO per side) and subcutaneous saline were injected. In another control group, intra-VTA vehicle (0.3μl 12% DMSO per side) was administered 5 min before the Reinstatement-test, and a subcutaneous morphine priming (1 mg/kg) was injected immediately before rats placed into the CPP chamber for the Reinstatement-test. The other three groups of rats received different bilateral doses of SB334867  in the VTA, 5 min before the reinstatement induced by a morphine priming . The animals were located into the CPP boxes for 10 min and permitted to move freely, and the CPP score and distance traveled were documented .2.9.At the end of the experiments, the rats were deeply anesthetized with ketamine and xylazine and perfused transcardially with a 0.9% saline solution followed by a solution of 10% formalin. Their brains were removed and stored in 10% formalin. The neuroanatomical location of cannulas tips were confirmed using the rat brain atlas . The dat2.10.The obtained data are presented as Mean±SD for each experimental group. To compare the conditioning scores or locomotor activity obtained in all control and experimental groups, 1-way Analysis of Variance (ANOVA) followed by post hoc analysis (Newman-Keuls) was used, as per appropriate. The statistical significance was set at P<0.05.3.3.1.11= 9.967, P<0.001) revealed that the animals exposed to the FSS, consumed more time on the morphine-paired side in comparison with NFSS group  indicated a significant difference between the FSS group and the animals that received two higher doses of SB334867  The Independent samples t-test result  presented that the rats that received an ineffective dose of morphine, consumed more time on the morphine-paired side in comparison with the saline-vehicle control group. It showed that an ineffective dose of morphine would induce significant reinstatement. To examine the effect of intra-VTA OX1R antagonist on the morphine priming-induced reinstatement, the animals received three doses of SB334867 microinjection into the VTA. The 1-way ANOVA followed by the Dunnett’s Multiple Comparison test result  indicated a significant difference between the morphine priming group which received vehicle and morphine priming groups which received the two higher doses of SB334867 in the reinstatement of CPP (1 and 3 nM/0.3 μL 12% DMSO per side)  indicated that priming dose of morphine and intra-VTA administration of the OX1R antagonist did not affect the locomotor activity  and Perifornical Area (PFA) but not in the Lateral Hypothalamus (LH) . GrowingWe propose that behavior responses contributing to FSS but not footshock regulate LH orexin-expressing neurons associated with the re ward system. Also, our results are almost intriguing with our previous findings, which indicated an indirect role of OX1R within DG in the reinstatement of FSS-induced morphine seeking . This diAnother conclusion might be drawn from this study is the prominent role of OX1R in morphine priming-induced reinstatement. Multiple lines of evidence have been demonstrated that activation of LH orexin neurons or intra-VTA administration of the orexin-A peptide reinstated morphine-seeking as measured by the CPP . In anotThe mechanism of orexin signaling during reinstatement of morphine seeking is unclear. Converging evidence reveals that the orexinergic system may regulate the activation of the mesolimbic dopamine system . MorphinOverall, we found that the activation of orexin transmission at OX1R in the VTA is directly implicated in the reward-seeking behavior induced by stress and morphine. We conclude that the orexin system may serve an important functional role in drug seeking and vulnerability to relapse. The orexin system may, therefore, be a target for preventing relapse during prolonged abstinence.